**3. Elucidating the mechanisms that lead to the production of IgG anti-lipid antibodies**

IgG anti-lipid antibodies have been detected in some infectious diseases, such as those caused by mycobacteria or in malaria, in individuals with autism-spectrum diseases and in individuals with autoimmune diseases, such as the anti-phospholipid syndrome and SLE [29, 30, 38, 39]. However, little is known about the mechanisms that lead to the production of these IgG antibodies. Understanding these mechanisms can help to understand the development of these diseases, and can lead to new therapeutic targets that improve the life quality of the patients. The mouse model of lupus induced by liposomes bearing NPA is particularly suitable to study the cellular and molecular mechanisms that lead to the production of IgG anti-lipid antibodies.

#### **3.1 Identification of anti-NPA antibody-producing plasma cells**

Plasma cells are the differentiation product of mature B cells [40]. Plasma cells can be short- or long-lived [41]. T-cell independent responses in the extrafollicular region of secondary lymphoid organs lead to the production of short-lived plasma cells, which in mice have a lifespan of less than a week. Long-lived plasma cells are generated in the germinal centers in T-cell dependent responses; after their generation, some of these cells migrate to the bone marrow and have a lifespan of months [42, 43].

Anti-NPA antibody-producing plasma cells were identified by flow cytometry as cells that contained intracellular NPA-bearing liposomes (stained with a lypophylic dye) and intracellular IgG antibodies [36]. These specific plasma cells were found in the spleen, the inguinal lymph nodes and the bone marrow of mice that produce

anti-NPA antibodies. A higher number of NPA-specific plasma cells were found in the spleen than in the lymph nodes [36], perhaps because the spleen contains additional B cell subsets (B1 and marginal zone B cells) that can also generate plasma cells [40, 44]. Interestingly, NPA-specific plasma cells were also found in the bone marrow [36]. Bone marrow plasma cells are generally considered to be long-lived, because they receive BAFF (B-cell activating factor) family cytokines, which promote their long-term survival. These cells can secrete low levels of antibodies for months or even years after the antigen is no longer present, to provide immediate protection if there is a subsequent encounter with the same antigen [42, 43].

#### **3.2 Determination of the NPA-specific B cell reaction pathway**

B cells respond *in vivo* via the germinal center pathway or via the extrafollicular reaction pathway [45, 46]. In germinal centers, isotype switching, affinity maturation and memory generation occur. These T cell-dependent processes can increase the affinity and the specificity of the antibodies, and B cells with high-affinity antibodies differentiate into plasma cells or memory B cells [31, 45]. On the other hand, the extrafollicular reaction leads to rapid production of low-affinity antibodies, which is sometimes associated with antibody class change [46, 47].

NPA-specific B cells were identified by flow cytometry as cells that bind to the NPA-bearing liposomes with their extracellular antibodies. Mice that received liposomes bearing NPA had abundant NPA-specific germinal center B cells, which increased over time, in their spleens and draining lymph nodes. In contrast, low numbers of NPA-specific extrafollicular B cells were found in the lymphoid organs of these mice. The affinity of the IgG anti-NPA antibodies produced by these mice increased over time, which further suggests that their B cells responded to NPA mainly through the germinal center pathway [36]. In order to access the germinal center pathway, B cells require T cell cooperation. However, conventional helper T cells are believed to respond only to protein antigens, so other T cell subsets may provide cooperation to the NPA-specific B cells found in this mouse model.

#### **3.3 Analysis of the cells that provide cooperation to the NPA-specific B cells**

NKT cells are a group of thymus-dependent T cells characterized by the expression of αβ TCR and several NK cell markers (NK 1.1, NKPR1 or CD161). Their development and functions are different from those of conventional CD4 and CD8 T cells. NKT cells cooperate with B cells that react with lipid antigens, and this cooperation leads to B cell activation, proliferation and differentiation into plasma cells [48, 49]. A subset of NKT cells, known as invariant NKT follicular helper (iNKTFH) cells, could provide cooperation to B cells, since their phenotype is similar to that of helper T cells [49]. These cells express CXCR5, Bcl-6 and CD40L, secrete cytokines and chemokines (IL-4, IFN-ɣ, IL-21 and BAFF) and are localized in germinal centers in response to the glycolipid α-galactosylceramide (α-GalCer) [50, 51] iNKTFH cells that are induced in response to other lipid antigens express CD40L, as occurs with follicular helper T cells (TFH), which are crucial for the development of germinal centers [52]. This evidence suggests that iNKTFH cells could provide cooperation to NPA-specific B cells, leading to their proliferation, affinity maturation and class-switching to IgG in a germinal center reaction. It remains to be determined if iNKTFH cells are indeed the cooperating T cell in this mouse model. Our proposed model for the generation of high-affinity IgG anti-NPA antibodies is shown in **Figure 5**.

*Anti-Non-Bilayer Phospholipid Arrangement Antibodies Trigger an Autoimmune Disease… DOI: http://dx.doi.org/10.5772/intechopen.106373*

#### **Figure 5.**

*Proposed mechanism for the production of anti-NPA antibodies. A naïve B cell recognizes the NPA antigen and responds. The B cell interacts with a helper T cell (possibly an iNKT cell) that induces its migration to a secondary lymphoid organ, in order to form a germinal center. In the germinal center, the B cell differentiates into a centroblast that proliferates and undergoes a series of mutations that generate low or high affinity antibodies. The centroblast then differentiates into a centrocyte, which no longer proliferates but undergoes antibody class-switching from IgM to IgG. B cells with high affinity antibodies are positively selected, while B cells with low affinity antibodies are negatively selected and die by apoptosis. When the B cell exits the germinal center, it differentiates into a memory B cell or a long-lived plasma cell that produces high affinity IgG anti-NPA antibodies. The B cells that do not receive T cell cooperation settle outside the follicles and differentiate into shortlived plasma cells that produce low-affinity IgM anti-NPA antibodies. Cell names in blue with a question mark indicate cells that have not yet been experimentally proven to participate in a germinal center specific to NPA.*
